Bonding of cartilaginous matrices using isolated chondrocytes

ABSTRACT

Methods of bonding cartilage pieces using new cartilage matrix generated by isolated chondrocytes in the presence of a biological gel. Also featured in the invention are cartilage implants used to repair cartilage defects.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisionalapplication Ser. No. 60/064,451, filed Oct. 30, 1997.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under NationalInstitutes of Health Grant AR31068.

FIELD OF THE INVENTION

[0003] The field of the invention is cartilage repair.

BACKGROUND OF THE INVENTION

[0004] Cartilage, which is a heterogeneous tissue, can be classified aseither articular or epiphyseal/physeal. Disturbances in cartilagestructure and function are seen in congenital, infectious, traumatic,degenerative and neoplastic conditions. Biological repair of focalarticular cartilage defects has generated great interest, yet some ofthe variables of this process have not been precisely defined (see,e.g., Brittberg et al., New Engl. J. Med., 331:889-895, 1994; andMankin, New Engl. J. Med., 331:940-941, 1994). Such variables includethe biochemical and biomechanical properties of the repair tissue itselfand also its bonding with adjacent cartilage and bone.

[0005] The morphogenetic scaffold to which chondrocytes may attach andform matrix is one of the variables that have effects on the repairtissue. Materials that have been used as the scaffold include collagengel (Fujisato et al., Biomaterials, 17:155-162, 1995; Hansen et al.,Clin. Orthop., 256:286-298, 1990; Mizuno et al., Exp. Cell. Res.,227:89-97, 1996; Nixon et al., Am. J. Vet. Res., 54:349-356, 1993; Samset al., Osteoarthr. Cartil., 3:47-59, 1995; Sams et al., Osteoarthr.Cartil., 3:61-70, 1995), fibrin glue (Hendrickson et al., J. Orthop.Res., 12:485-497, 1994; Homminga et al., Acta Ortopedica Scandinavica,64:441-445, 1993; Tsai et al., J. Formosan Med. Assoc., 3(Suppl):239-245, 1993), polyglycolic acid (Freed et al., Biotechnology,12:689-693, 1994; Vacanti et al., Am. J. Sports Med., 22:485-488, 1994),polyethylene oxide gel (Sims et al., Plast. Reconstr. Surgery,98:843-850, 1996), alginate gel (Van Susante et al., Acta OrtopedicaScandinavica, 66:549-556, 1995), carbon fiber pads (Brittberg et al.,Clin. Orthop., 326:270-283, 1996) and xenogeneic matrix (Caruso et al.,J. Orthop. Res., 14:102-107, 1996).

[0006] Isolated and cultured chondrocytes embedded in these variousscaffolds have been used for filling and repairing articular cartilagedefects in chicks (Itay et al., Clin. Orthop., 220:284-303, 1987),rabbits (Grande et al., Anatomical Records, 218:142-148, 1987; Grande etal., J. Orthop. Res. 7:208-218, 1989; Wakitani et al., J. Bone JointSurg. [Br.], 71:74-80, 1989), dogs (Shortkroff et al., Biomaterials,17:147-154, 1996), and horses (Hendrickson et al., supra; Sams et al.,Osteoarthr. Cartil., 3:47-59, 1995; Sams et al., Osteoarthr. Cartil.,3:61-70, 1995).

[0007] Complete repair of partial defects of cartilage impliesside-to-side joining of cartilaginous matrices. While such joining hasbeen investigated in several ways (Hunziker et al., Trans. Orthop. Res.Soc., 17:231, 1992; Reindel et al., J. Orthop. Res., 13:751-760, 1995;Wolohan et al., J. Orthop. Res., 9:180-185, 1991), options foraccomplishing this still need to be expanded.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the discovery that isolatedchondrocytes can propagate in the presence of an appropriate biologicalgel (e.g., fibrin gel) and generate cartilage matrix that firmly bondstwo adjacent cartilage pieces.

[0009] Accordingly, the invention features a method of bonding twocartilage pieces. In this method, a bonding composition containingisolated chondrocytes mixed with a biological gel is applied to asurface of one (or both) of the cartilage pieces, and the surface isthen contacted with the other cartilage piece. New cartilage matrixgenerated by the bonding composition will provide durable (e.g., 1% ormore of normal cartilage strength) bonding between the two contactingcartilage pieces. The order of steps in the above-described method canbe altered. For instance, the two cartilage pieces to be bonded can beheld in apposition, and then the bonding composition is applied to fillgaps at the interface of the two cartilage pieces.

[0010] In another method of the present invention, either or both of thecartilage pieces are first incubated with isolated chondrocytes. Abiological gel is then applied to a surface of either or both of the twopieces, and the two pieces are held together at the surface.Alternatively, the two cartilage pieces to be bonded can be held inapposition first to form a cartilage composite; after the composite isincubated with isolated chondrocytes, a biological gel is applied tofill gaps at the interface of the two cartilage pieces.

[0011] Isolated chondrocytes are chondrocytes that are separated fromcartilage matrix, and they can be obtained from cartilage tissue or bonemarrow. Both freshly isolated and cultured chondrocytes can be used.

[0012] A biological gel is a flexible, biodegradable (i.e.,bioresorbable) and biocompatible (i.e., has no or negligible in vivotoxicity and is compatible with in vivo conditions) composition thattypically has pores large enough to allow chondrocytes to populate. Anexemplary biological gel is fibrin gel (also called fibrin glue). Fibringel has been used as the basis of many biological glues or adhesivematrices, and can be prepared with coagulation factors includingthrombin and fibrinogen. Fibrinogen is cleaved by thrombin to formfibrin at the initiation of clotting.

[0013] One or both of the cartilage pieces to be bonded can be depletedof endogenous (i.e., innate) chondrocytes before the isolatedchondrocytes are applied. The cartilage pieces to be bonded can bearticular cartilage, fibrocartilage or growth cartilage, and can beobtained from the patient to be treated, or from a donor of the same ordifferent species.

[0014] The new methods can be used to repair (i.e., resurface), in amammal (e.g., human, mouse, rat, dog, horse, lamb, sheep, etc.),articular cartilage having a defect (e.g., a partial or full thicknessdefect); in that case, one of the two pieces to be bonded is thedefective cartilage, and the other piece constitutes a part of acartilage implant, and the chondrocytes used can be derived from themammal itself. The present methods can also be used to treat defects inother types of cartilage, e.g., a meniscal tear in fibrocartilage or aresection defect resulting from excision of a physeal bar from aninjured growth plate.

[0015] Also featured in the invention is a method of preparing acartilage implant. In this method, a cartilage piece (e.g., one thatcontains no or essentially no viable endogenous chondrocytes) ofappropriate size and shape is first co-cultured with isolatedchondrocytes, and then a biological gel is applied to the cartilagepiece to generate an implant. Alternatively, a bonding composition asdescribed above can be applied directly the cartilage piece to generatean implant.

[0016] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples described herein are illustrative only and notintended to be limiting.

[0017] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A and 1B are graphs showing applied tensile displacementand measured tensile load, respectively, from an experimental cartilagecomposite seeded with isolated chondrocytes and implanted in vivo for 21days.

[0019]FIG. 1C is a graph showing a stress-strain curve generated fromdata shown in FIGS. 1A and 1B by normalizing displacement data tomeasured sample thickness and load data to sample area. S_(UTS) standsfor ultimate tensile strength, ε_(f) for fracture strain, M for dynamictensile modulus, and E_(f) for fracture energy.

[0020] FIGS. 2A-2D are graphs showing the time course of changes intensile strength, fracture strain, fracture energy, and tensile modulus,respectively, of experimental and control cartilage composites. Thecomposites were implanted in nude mice for the time period indicated onthe horizontal axis. All data are shown as mean±SD, with the number ofdata points at each time point ranging from 4 to 6 for experimentalcomposites and 2 to 4 for controls. In each of the four figures, “*”denotes appropriate p value for significance of difference between theexperimental group and the control group at that time point.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The methods of the invention are useful for bonding two or morecartilage pieces. In one of these methods, one first obtains a bondingcomposition comprising isolated chondrocytes mixed with a biological gel(e.g., about 10⁵ to 10⁷ cells/per ml gel), and then applies the bondingcomposition to the interface of the two cartilage pieces. Alternatively,one can first co-culture one or both of the cartilage pieces (e.g.,devitalized pieces) with isolated chondrocytes (at a concentration ofabout 10⁵ to 10⁷ cells/per ml medium), and then apply a biological gelto the interface of the pieces. The chondrocytes that have infiltratedthe cartilage pieces will then migrate into the gel.

[0022] The biological gel serves as a biodegradable and biocompatiblescaffold on which the chondrocytes will proliferate and generate durablecartilage matrix. Biological gels that may be used include, but are notlimited to, collagen gel, fibrin glue, polyglycolic acid, polylacticacid, polyethylene oxide gel, alginate or calcium alginate gel,poly-(2-hydroxyethyl methacrylate) (i.e., a hydrogel), polyorthoester,hyaluronic acid, polyanhydride, gelatin, agarose, and otherbioresorbable and biocompatible materials such as those described in EP0705878 A2. To promote chondrocyte proliferation and function, thebiological gel can additionally contain appropriate nutrients (e.g.,serum, salts such as calcium chloride, ascorbic acid, and amino acids)and growth factors (e.g., somatomedin, basic fibroblast growth factor,transforming growth factor β, cartilage growth factor, bone-derivedgrowth factor, or a combination thereof). Selection of the optimalbiological gel can be made using the guidance provided in the Examplesbelow.

[0023] Chondrocytes useful in the new methods can be isolated from,e.g., articular cartilage or epiphysial growth-plate, by digestion withcollagenase and optionally trypsin. Mesenchymal cells obtained from bonemarrow can also be differentiated into chondrocytes under appropriateculture conditions as described by, e.g., Butnariu-Ephrat et al.,Clinical Orthopaedics and Related Research, 330:234-243, 1996. Othersources from which chondrocytes can be derived include dermal cells andpluripotent stem cells.

[0024] The methods of the invention can be used to repair defectivearticular cartilage. To do so, a cartilage piece cut into the shape andsize of a defect (see, e.g., Chu et al., Arch. Am. Acad. Orthop. Surg.,1:9-14, 1997) is press-fitted and bonded to the defect. The cartilageimplant can be, for example, autogeneic, isogeneic (e.g., from anidentical twin), allogeneic, or xenogeneic. Before new cartilage matrixis generated at the interface of the implant and the host bed, resultingin a durable bonding between the implant and the host bed, the implantcan be held in place by, e.g., bioresorbable pins (see, e.g., Chu etal., supra), or a piece of periosteal/perichondrial tissue sutured overthe site of implantation (see, e.g., Minas et al., Orthopedics,20:525-538, 1997; and WO 97/30662). One can also select an adhesivebiological gel (e.g., fibrin gel) that provides temporary adhesionbetween the two cartilage pieces. The chondrocytes used for repairingcartilage defects can be, e.g., autogeneic, isogeneic, or allogeneic.Before implantation, the host cartilage to be repaired can be treatedwith, e.g., an enzyme, to remove proteoglycans and other substances thatmay interfere with the bonding.

[0025] The following examples are meant to illustrate the methods andmaterials of the present invention. Suitable modifications andadaptations of the variety of conditions and parameters normallyencountered in bonding cartilaginous matrices which are obvious to thoseskilled in the art are within the spirit and scope of the presentinvention.

EXAMPLE I: Preparation of Cartilage Implant MATERIALS AND METHODS

[0026] Chondrocyte Isolation

[0027] Articular cartilage was harvested from lamb hips and shoulders byremoving the superficial layers of articular cartilage under sterileconditions. The subchondral bone was avoided. Cartilage pieces wereincubated for 8 hours at 37° C. in HAM's medium with Glutamax-1(GibcoBRL, Grand Island, N.Y.) containing 0.075% collagenase Type 2(Worthington Biochemical Co. Freehold, N.J.), 10% fetal bovine serum(Sigma, St. Louis, Mo.), 50 μg/ml ascorbic acid, 1%antibiotic/antimycotic solution (Sigma; each ml of the solution contains10,000 units Penicillin, 10 mg Streptomycin, and 25 μg Amphotericin B in0.9% NaCl), and L-glutamine 292 mg/l. Subsequent to the incubation,undigested tissue and debris were removed by filtering the cellsuspension through a sterile nylon gauze.

[0028] Processing of Allogeneic Matrix

[0029] Slices of articular cartilage, each measuring approximately 3millimeters (“mm”) in width, 5 mm in length, and 1 mm in thickness, wereharvested from knees and shoulders of unrelated lambs under sterileconditions. They were then placed in 50 ml test tubes containingphosphate buffered saline (“PBS”) and 2% of the antibiotic/antimycoticsolution (Sigma) and frozen at minus 20° C. for five days. Afterthawing, the PBS solution was discarded and the cartilage slices weresubjected to five cycles of freezing and thawing in the absence of PBS.

[0030] Next, chondrocyte viability was evaluated in exemplary pieces byTrypan blue staining (Trypan blue 0.2%, Sigma) and fluorescencemicroscopy as previously described (Vacanti et al., supra). Thisprocedure revealed that the cyclic freezing and thawing had killed allthe innate chondrocytes in the cartilage slices. Cartilage slices withno detectable chondrocytes were termed “non-viable cartilage matrix” or“devitalized cartilage matrix.”

[0031] Co-Culturing of Chondrocytes with Allogeneic Matrix

[0032] The cell suspension obtained as described above was centrifugedat 4000 rpm for ten minutes, and washed in PBS containing 2% of theantibiotic/antimycotic solution. Viability of chondrocytes was assessedby Trypan blue staining and recorded as a percentage of viablechondrocytes per high power field. Only those chondrocyte cultureshaving a viability score of 90% or greater were used in further studiesdescribed below. The exact cell count per ml was established using ahemocytometer. Chondrocyte solutions were adjusted to a concentration of10⁶ cells/ml prior to use.

[0033] Co-culturing of chondrocytes with allogeneic matrix was performedby placing three slices of non-viable cartilage matrix in a 12 ml testtube (Corning, N.Y., USA) containing 4 ml of F12 medium (Sigma) and 1 mlof the adjusted chondrocyte solution. The cartilage slices and thechondrocytes were co-cultured in the F12 medium for 21 days. Controlsamples that contained no chondrocytes were kept under the same culturecondition. In all cultures, the medium was changed twice weekly. Forexperimental groups, the fresh medium contained 10⁶ chondrocytes/ml.

[0034] Preparation of Matrix Composites

[0035] After co-culturing, the three cartilage slices infiltrated withchondrocytes (i.e., experimental slices) were removed from the media.This was done by placing the test tube on a vortex machine to re-suspendchondrocytes that were only loosely attached to the cartilage slices.The medium was then removed by aspiration, and the cartilage slices werere-suspended in fresh medium.

[0036] After this washing procedure was repeated several times, thethree cartilage slices were placed onto a sterile Petri dish, andstacked on a sterile 27 gauge insulin needle by piercing each onethrough the center. Then, fibrin glue gel made from humancryoprecipitate and bovine thrombin (USP-Thrombostat, Parke DavisLambert Co, Morris Plains, N.J.) was applied around the three slices toform a composite cartilage unit. The fibrin glue set in a few minutes,and the needle was subsequently removed.

[0037] Control composites were made with non-viable cartilage slicesthat had not been co-cultured with isolated chondrocytes.

[0038] Animal Model

[0039] Implantation of experimental and control matrix composites wasperformed under sterile conditions in a laminar flow hood. Thecomposites were implanted into subcutaneous pouches at four sites in thebacks of nude mice. Two experimental and two control composites wereimplanted in each animal. After sacrifice of the animals, cartilagecomposites were recovered under sterile conditions and evaluated asdescribed below.

[0040] Evaluation of Composites Recovered from Mice

[0041] As shown in Table 1, three separate animal groups were employedfor three different studies.

[0042] The first group, which consisted of 4 mice (each with fourcomposites), was used to assess the bonding and histology of the matrixcomposites at various times (i.e., at 7, 14, 21, 28 and 42 daysfollowing implantation). The composites were examined with a pair ofjeweler's forceps for the existence of fusion at the contact planesbetween the adjacent cartilage slices and separatability of the slicesby the forceps' opening force. Each composite of three cartilage sliceshad two contact planes. The data on bonding between the cartilage slicespresented below and in Table 2 refer to the 16 contact planes in the 8experimental cartilage composites and to the 16 contact planes in the 8control cartilage composites for each time period. A rank order scaleused to record the bonding between the cartilage slices. When thecartilage slices were completely fused with one another at their contactplanes, a value of one was assigned. When any separation at all betweenslices was produced by the forceps, a value of zero was assigned.Bonding was expressed in absolute numbers and percentages per group.Composites used for histological analysis were fixed in 10% phosphatebuffered formalin, embedded in paraffin, sectioned at five micrometers,stained with Safranin-O, and examined under a light microscope at 200×or 400×. TABLE 1 Time periods: Day 0 (not Study group implanted) Day 7Day 14 Day 21 Day 28 Day 42 Totals Number of Experimental  8 8  8 8  8 8 48 implants for [4]  [4] [4]  [4]  [4] [20] surfaces bonding Control 8 8  8 8  8  8 48 evaluation and histologic analysis Number ofExperimental  8 —  8 —  8  8 32 implants for  [4]  [4]  [4] [12] [³H]Thymidine Control  8 —  8 —  8  8 32 incorporation analysis Number ofExperimental  8 —  8 —  8  8 32 implants for  [4]  [4]  [4] [12]fluorescence Control  8 —  8 —  8  8 32 microscopy evaluation Totals:Experimental 24 8 24 8 24 24 88 [4] [12] [4] [12] [12] [44] Control 24 824 8 24 24 88

[0043] TABLE 2 Experimental group Control group DAY Bonding Bonding 00/16 (0%) 0/16 (0%) 7 0/16 (0%) 0/16 (0%) 14 4/16 (25%) 0/16 (0%) 2112/16 (75%) 0/16 (0%) 28 16/16 (100%) 0/16 (0%) 42 16/16 (100%) 0/16(0%)

[0044] The second group, which consisted of 12 mice (each with fourcomposites), was divided evenly into 3 subgroups for evaluatingchondrocyte division at 14, 28, and 42 days, respectively, followingimplantation surgery. To evaluate chondrocyte division, each recoveredcomposite was incubated with 16 μCi/ml of [³H]thymidine for 24 hours inan atmosphere of 92% air and 8% C₂. Each composite was then hydrolyzedand combined with 3 ml of CYTOSCINT™ (ICN, Costa Mesa, Calif.).Radioactivity counts released from the composite were determined with aBECKMAN LS5000TD β-scintillation counter (Beckman, Fullerton, Calif.).

[0045] The third animal group consisted of 12 mice as well, and was usedto evaluate composites recovered therefrom by fluorescence microscopy.Fifteen 100 μm thick sections of experimental and control cartilagecomposites were prepared using a 752M VIBROSLICE™ microtome (CampdenInstruments LTD, Loughborough, England). These sections were thenincubated with 100 μl of fluorescent dye solution (consisting of 3 μlcalcein AM and 8 μl ethidium homodimer in 5 ml PBS; Molecular Probes,Eugene, Oreg.) for 1 hour. Cell viability within the matrix ofexperimental and control cartilage composite units was assessed on fresh100 μm thick sections using a fluorescent microscope (NikonMICROPHOT-FX™, Garden City, N.Y.). The calcein is taken up by viablecells and strongly fluoresces green, which is seen as a region of highlight intensity on black and white photographs. The ethidium homodimerpassively enters non-viable cells and weakly fluoresces red, which isseen as a region of low light intensity on black and white photographs.

[0046] Baseline values on composites prior to implantation were obtainedfor bonding (0/16), histology, [³H]thymidine incorporation andfluorescence.

[0047] Statistical Analysis

[0048] Unpaired Student's t tests were used to compare [³H]thymidineincorporation values (mean±SD) collected throughout the time periodsexamined. The Bonferroni modification was employed to maintain a type Ierror rate of 0.05 across all comparisons. In addition, experimental andcontrol values during each session of the study were compared by meansof unpaired Student's t tests (p<0.05).

RESULTS

[0049] Starting at day 7 after implantation, fibrous capsules wereformed by the fibrin glue previously applied to hold the piecestogether, and they thickened with time. The capsules were removed forevaluation of bonding. The bonding of the experimental matricesinfiltrated with viable chondrocytes increased with time until all suchspecimens were united at approximately days 28 to 42. Macroscopic viewof the experimental composites at 42 days following implantationrevealed that, while the original slices of matrix were discernible, thecomposite had united into a solid cartilaginous mass. However, at alltime points examined, the cartilage pieces in the control compositesslid apart spontaneously and immediately after the removal of thefibrous capsule. Thus, the distinction between success and failure inbonding was easy to discern.

[0050] Histological evaluation showed that, prior to implantation, theexperimental composites had live chondrocytes on the surface of theirdevitalized cartilage slices, whereas only some nuclear debris of deadcells but no vital cells were found in control composites.

[0051] Microscopic examination of cartilage composites recovered afterimplantation revealed that chondrocytes were forming matrix at thecontact planes between the allogeneic slices of experimental composites,and that this new cartilage layer increased in thickness from day 7 today 42. At day 7 after implantation, viable chondrocytes forming matrixwere seen on the contacting surfaces of cartilage slices, and the fibringlue formed a relatively thick layer between the contact surfaces. Atday 14, each layer of viable chondrocytes had increased in thickness,and the fibrin glue layer had shrunk. By day 21, the contact spacebetween the elliptically shaped allogeneic matrices was filled withviable chondrocytes making new matrix, and the fibrin glue had beenmostly absorbed. 28 days following implantation, the entire contactingregion between adjacent cartilage slices was filled with new cartilage.Buds of new cartilage started to grow into the devitalized matrices. Atday 42, more ingrowth of the buds, some of which had branches, was seen,and loss of Safranin-O staining of the devitalized matrix occurredaround the new cartilage, indicating that matrix-generating chondrocyteswere invading the devitalized matrix. By this time, new cartilage, theorganization of which grossly resembled that of mature cartilage, hadentirely filled the holes in the cartilage slices created by the needleused during composite assembly with fibrin glue. Safranin-O staining inthe new cartilage had also increased. Several mitotic figures wereencountered in the new cartilage layer at every time period.

[0052] The control composites, on the other hand, showed no new matrixformation at any time point examined. Sectioning of these compositescould be accomplished only because of the surrounding fibrous capsule.The capsule stained only with the Fast Green counter-stain and notSafranin-0. No viable chondrocytes were found present.

[0053] Examination of chondrocyte division showed a statisticallysignificant decrease in [³H]thymidine incorporation into theexperimental composites from day 0 (about 85,000 counts per minute orcpm) to day 28 (about 10,000 cpm), followed by an increase at day 42(about 43,000 cpm). The differences at the various time points for theexperimental composites were significant, with p less than 0.01 inStudent' t test. Incorporation of [³H]thymidine into control compositeswas not significantly different from baseline (about 2,000 cpm) at anytime period. The differences between the experimental and control groupswere significant (p<0.05) at each time point examined (i.e., days 0, 14,28, and 42).

[0054] Examination of the experimental composites with fluorescencemicroscopy (at 10OX) confirmed that the growth of the new matrix wasoccurring with a concomitant increase in viable chondrocytesinterstitially. Fluorescence staining of experimental composites at day42 revealed that the devitalized matrices did not take up calcein,whereas the new cartilage layer formed between neighboring matricesfluoresced brightly with calcein, indicating the presence of viablecells in the layer. Buds of new cartilage that fluoresced brightly withcalcein were also seen penetrating the devitalized matrix. Viableclusters of chondrocytes were seen within the allogeneic matrices atdays 28 and 42.

EXAMPLE II: Biomechanical Assessment of Cartilage Bonding

[0055] To test the strength of the bonding formed between two cartilagedisks as described in the above Example, the pair of bonded disks wereglued onto plexiglass rods and mounted in the jaws of a testing machine.The bonded slices were pulled to failure in tension as indicated byeither visible separation of cartilage disks or when the measured loadwas observed to be less than 0.05 Newton. Resultant loads were recordedon a personal computer, and data were collected at a rate of 5points/second. Sample pairs were kept hydrated with PBS throughouttesting.

[0056] Applied displacements and measured loads were normalized tosample thickness and area, and using these data, a stress-strain curvewas constructed for each sample. From the stress-strain curve, theultimate tensile strength (S_(UTS)) was determined by inspection as thestrain at which further increases in strain produced lower stresses. Thefracture strain (ε_(f)) was also determined by inspection. The fractureenergy (E_(f)), defined as the area under the stress-strain curve untilfailure, was calculated numerically using a Reimann sum method with thepartition element given by the strain interval between data points. Thedynamic tensile modulus (M) for the given strain rate of each sample wascalculated as the slope of the linear portion of the stress-strain curveusing a standard least squares algorithm.

[0057] Cartilage composites implanted in nude mice as described inExample I were retrieved at a weekly interval for up to 6 weeks. Tensilestrength, fracture strain, fracture energy, and tensile modulus of thecartilage bonding formed in these composites were assessed. The valuesof these parameters are indicative of bonding strength. Data shown inFIGS. 2A-2D demonstrated that the values of the four parameters weresignificantly higher in cartilage composites containing isolatedchondrocytes than in composites not containing chondrocytes. The valuesalso increased steadily with time in the former composites, and by week6, reached 5-10% of the corresponding values of normal articularcartilage.

[0058] After biomechanical tests, samples were retrieved forhistological evaluation. Histological evaluation was also performed on afew samples that were not biomechanically tested. For pre- and post-testhistological analysis, specimens were fixed in 10% phosphate bufferedformalin and embedded in paraffin. Serial sections that were 5 μM thickwere obtained and stained with Safranin-O.

[0059] Histological analysis of biomechanically tested samples indicatedthat failure of bonding occurred at the interface between the new anddevitalized cartilage in experimental groups or between the two discs ofcartilage for controls. After the two layer of new matrix fused at aboutday 21, failure was never observed in the thickness of the new tissue,but always at the interface of the newly formed matrix and thedevitalized cartilage.

[0060] In samples fixed during testing, newly formed tissue was pulledaway from devitalized cartilage matrix. This was analogous to anapparent crack propagating along the interface between new anddevitalized matrix. Observation of this process revealed that, invicinity of the crack tip, cells along the fracture line appeared to beelongated in the direction perpendicular to the line of crackpropagation. Occasionally, fractures occurred in opposite sides and bothpropagated towards the center, leaving an intact connection.

[0061] In case where chondrocytes penetrated devitalized matrix,fracture occurred at the opposing interface in 80% of the sectionssampled.

[0062] As shown above, the strength of the interface between the newtissue and existing matrix increases with time, demonstratingremodelling of the new tissue, even after the space between the piecesof matrix has been filled with new tissue. Although the total fusion ofthe two layers of new matrix occurred at about day 21, as shown by thehistology, the biomechanical study showed an increase of the strength ofthe repair tissue at further evaluation times (days 28 and 42). Thisphenomenon could be explained by the formation of buds of penetrationfrom the new tissue into the devitalized matrix, stabilizing theconstruct and making the interface stronger; this is consistent withhistological finding that in 80% of sections sampled, failure occurredon the opposite interface when penetration were present.

EXAMPLE III: Meniscus Repair

[0063] This example applies the cartilage repair procedure of Example Ito meniscus repair.

[0064] Articular cartilage was harvested from lamb joints, and thechondrocytes isolated as described in Example I above. Meniscus chipswere harvested from the knees of lambs of the same species.

[0065] Menisci were harvested from unrelated lambs, and each meniscuswas divided into three parts. The vascularized portion was removed fromthe avascular zone of each part, and the menisci were devitalized usingfive freeze/thaw cycles. A 4 mm-long buckle handle lesion (also called afracture) was carved into each meniscus about 2 mm from the free marginof the avascular zone.

[0066] The devitalized chips were co-cultured with lamb chondrocytes asdescribed in Example I. The menisci were divided into four groups. Ingroup A, a chip seeded with chondrocytes was sutured inside the bucklehandle fracture of the meniscus. In group B, an unseeded chip wassutured onto the fracture. In group C, the meniscus fracture was suturedwithout a chip. In group D, the fracture was left untreated (nosuturing).

[0067] The fibrin glue gel described in Example I was then applied tothe meniscus samples in each group so as to surround the samples withthe gel. Each of two samples from each group was implanted into thesubcutaneous pouch of a nude mouse, totalling eight mice in all.

[0068] After 14 weeks, the meniscus samples were removed from the mice,grossly and histologically examined, and tested for cartilage repair asdescribed in Example I. The samples from group A kept their shape andwere fully repaired, while the samples from groups B-D did not indicateany repair.

EXAMPLE IV: Clinical Repair of Articular Cartilage

[0069] This example describes a prophetic protocol (adapted from Chu etal., supra) for repairing a full thickness articular cartilage defect ina knee joint of a human.

[0070] The surgical procedure entails entry of the knee joint through astandard midline incision. The damaged articular surface is removed withan osteotome in a rectangular pattern. Approximately 5 mm of subchondralbone is removed with a high-speed burr. The host bed is then measured.

[0071] A similarly sized and located allograft is removed from a freshcadaver knee of a healthy donor. The cadaver knee matches the defectiveknee in size, as determined by the anteroposterior dimension of thetibial plateau on standard radiographs. The allograft subchondral boneis tailored with a burr to a thickness of 5 to 10 mm. Pulsatile lavageis used to flush out cellular elements from the marrow. A bondingcomposition containing fibrin gel and chondrocytes derived from the bonemarrow of the patient (about 10⁶ cells/ml) is applied to the host bedand the allograft at places where the two will contact. The allograft isthen press-fitted into the host bed and positioned about 1 to 2 mm abovethe articular surface of the host bone. Resorbable pins are used fortemporary internal fixation.

Other embodiments

[0072] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims:

[0073] Other aspects, advantages, and modifications are within the scopeof the following claims.

What is claimed is:
 1. A method of bonding a first cartilage piece and asecond cartilage piece, the method comprising: providing a bondingcomposition comprising isolated chondrocytes mixed with a biologicalgel; applying the composition to a surface of the first cartilage piece;and contacting the surface with the second cartilage piece.
 2. Themethod of claim 1 , wherein the biological gel is fibrin gel.
 3. Themethod of claim 1 , wherein the first cartilage piece contains no viableendogenous chondrocytes.
 4. The method of claim 1 , wherein the secondcartilage piece is a defective part of a joint in a mammal.
 5. Themethod of claim 4 , wherein the biological gel is fibrin gel.
 6. Amethod of bonding a first cartilage piece and a second cartilage piece,the method comprising: providing a bonding composition comprisingisolated chondrocytes mixed with a biological gel; holding the twocartilage pieces in apposition; and filling gaps between the twocartilage pieces with the bonding composition.
 7. The method of claim 6, wherein the biological gel is fibrin gel.
 8. The method of claim 6 ,wherein the first cartilage piece contains no viable endogenouschondrocytes.
 9. The method of claim 6 , wherein the second cartilagepiece is a defective part of a joint in a mammal.
 10. The method ofclaim 9 , wherein the biological gel is fibrin gel.
 11. A method ofbonding a first cartilage piece and a second cartilage piece, the methodcomprising: providing isolated chondrocytes; contacting the chondrocyteswith either or both of the cartilage pieces; applying a biological gelto a surface of the first cartilage piece; and contacting the surfacewith the second cartilage piece, in the presence of the chondrocytes.12. The method of claim 11 , wherein the biological gel is fibrin gel.13. The method of claim 11 , wherein the cartilage piece that iscontacted with the isolated chondrocytes contains no viable endogenouschondrocytes.
 14. The method of claim 11 , wherein the second cartilagepiece is a defective part of a joint in a mammal.
 15. The method ofclaim 14 , wherein the biological gel is fibrin gel.
 16. A method ofbonding a first cartilage piece and a second cartilage piece, the methodcomprising: providing isolated chondrocytes; contacting the chondrocyteswith either or both of the cartilage pieces; holding the two cartilagepieces in apposition; and filling gaps between the two cartilage pieceswith a biological gel.
 17. The method of claim 16 , wherein thebiological gel is fibrin gel.
 18. The method of claim 16 , wherein thecartilage piece that is contacted with the isolated chondrocytescontains no viable endogenous chondrocytes.
 19. The method of claim 16 ,wherein the second cartilage piece is a defective part of a joint in amammal.
 20. The method of claim 19 , wherein the biological gel isfibrin gel.
 21. A method of preparing a cartilage implant, the methodcomprising: providing isolated chondrocytes and a cartilage piece;incubating the chondrocytes with the cartilage piece to produce anincubated cartilage piece; and applying a biological gel to theincubated cartilage piece to generate a cartilage implant.
 22. Themethod of claim 21 , wherein the biological gel is fibrin gel.
 23. Themethod of claim 21 , wherein the cartilage piece contains no viableendogenous chondrocytes.
 24. The method of claim 22 , wherein thecartilage piece is obtained from a mammal.
 25. A cartilage implantcomprising a cartilage piece and a bonding composition on a surface ofthe cartilage piece, the bonding composition containing a biological geland isolated chondrocytes.
 26. A cartilage implant comprising acartilage piece, a biological gel on a surface of the cartilage piece,and chondrocytes exogenously introduced to the cartilage piece.